Marijuana is currently the most widely abused illegal drug. However, the functional significance of the cannabinoid receptor system in health and disease includes the use of cannabinoids as analgesics, antiemetics in cancer patients, anticonvulsants for epilepsy and as antiglaucoma agents as well as immunomodulatory agents. To date, two cannabinoid receptor subtypes, CBt and CB2, have been identified by cDNA cloning, yet the complex pharmacological properties of anandamide as well as exogenous cannabinoids are not fully defined by these two subtypes. Anandamide produces the full range of behavioral effects (antinociception, catalepsy and impaired locomotor activity) in CBi receptor knockout mice. In addition, anandamide-stimulated GTPyS activity can be elicited in brain membranes from these mice. Finally, radioligand binding studies indicate the existence of additional binding sites in brain and spinal cord. The goal of this project is to identify and characterize additional subtypes of the cannabinoid receptors in order to elucidate their role in vivo. The first specific aim is to characterize candidate orphan GPCRs for cannabinoid receptor activity. GPR55 has recently been identified as a candidate cannabinoid receptor, and we have evidence that GPR35 has cannabinoid receptor activity. GPR18 is a receptor for Narachinoylglycine, an endocannabinoid. GPR119 is a receptor for oleoylethanolamide and palmitoylethanolamide. We propose to characterize these candidate cannabinoid receptors by expression in heterologous cell lines, allowing the detailed characterization of ligand selectivity and signaling. The second specific aim will identify additional orphan GPCRs with cannabinoid receptor activity using an expression cloning strategy. In specific aim 3, we will investigate the functional role of novel cannabinoid receptors. The experiments in this aim are designed to utilize our collaborations with other Center Grant Investigators in order to understand the functional roles of the candidate cannabinoid receptors. These studies will facilitate the design of new therapeutic strategies involving the cannabinoid system. Furthermore, insight into the molecular mechanisms of activation of cannabinoid receptors may lead to a better understanding of marijuana abuse in humans.